US 12,215,370 B2
Methods for detecting target analytes using synthetic biological circuits that modify glucose levels in a cell-free system
Keith Pardee, Toronto (CA); Evan Amalfitano, Oakville (CA); and Margot Karlikow, Toronto (CA)
Assigned to The Governing Council of the University of Toronto, Toronto (CA)
Appl. No. 16/956,431
Filed by The Governing Council of the University of Toronto, Toronto (CA)
PCT Filed Dec. 21, 2018, PCT No. PCT/CA2018/051646
§ 371(c)(1), (2) Date Jun. 19, 2020,
PCT Pub. No. WO2019/119148, PCT Pub. Date Jun. 27, 2019.
Claims priority of provisional application 62/609,525, filed on Dec. 22, 2017.
Prior Publication US 2020/0318148 A1, Oct. 8, 2020
Int. Cl. C12P 19/02 (2006.01); C12P 19/14 (2006.01); C12Q 1/54 (2006.01)
CPC C12P 19/02 (2013.01) [C12P 19/14 (2013.01); C12Q 1/54 (2013.01); C12Y 101/01047 (2013.01); C12Y 301/03009 (2013.01); C12Y 302/01001 (2013.01); C12Y 302/0102 (2013.01); C12Y 302/01021 (2013.01); C12Y 302/01023 (2013.01); C12Y 302/01026 (2013.01); C12Y 302/01028 (2013.01)] 5 Claims
 
1. A method for modifying glucose in response to a target nucleic acid molecule in a sample, the method comprising:
(i) contacting the sample with a synthetic biological circuit within a cell-free system reaction volume, the reaction volume present in a container, wherein the synthetic biological circuit comprises a DNA molecule comprising a promoter operably linked to a DNA molecule encoding an enzyme whose expression modifies the level of glucose in the reaction volume and the target nucleic acid molecule activates the synthetic biological circuit in the reaction volume to regulate translation of a mRNA molecule encoding the enzyme to modify the level of glucose in the reaction volume, and
(ii) detecting glucose in the cell-free system reaction volume using a glucose meter thereby detecting the target nucleic acid molecule in the sample,
wherein the synthetic biological circuit further comprises a riboregulator that controls translation of the mRNA molecule encoding the enzyme, wherein the riboregulator recognizes the target nucleic acid molecule and the target nucleic acid molecule is a trigger for the riboregulator such that binding of the target nucleic acid molecule to the riboregulator changes the conformation of the riboregulator thereby permitting translation of the mRNA encoding the enzyme in the synthetic biological circuit, and
wherein the cell-free system comprises a substrate that is acted on by the enzyme to generate glucose and
i) the substrate is trehalose and the enzyme is trehalase;
ii) the substrate is maltose and the enzyme is maltase;
iii) the substrate is cellobiose and the enzyme is cellobiase;
iv) the substrate is starch and the enzyme is amylase;
v) the substrate is lactose and the enzyme is lactase (beta-galactosidase);
vi) the substrate is sucrose and the enzyme is invertase or sucrase;
vii) the substrate is glucose and the enzyme is glucose dehydrogenase; or
viii) the substrate is glucose-6-phosphate or glucose-1-phosphate and the enzyme is glucose 6-phosphatase.